Fibroblast Growth Factor Receptor-Derived Peptides Binding to NCAM

ABSTRACT

The present invention relates to the use of peptides that are capable of binding to, and modulating the activity of NCAM. The peptides are peptide fragments of FGFRs. They are derived from two distinct binding sites for binding of the immunoglobulin-like module 2 of FGFR to NCAM F3 modules 1-2. The invention further relates to use of said peptides for the production of a medicament for the treatment of different pathological conditions, wherein NCAM and/or FGFRs play a prominent role.

FIELD OF INVENTION

The present invention relates to the use of peptides that are capable ofbinding to, and modulating the activity of NCAM. The peptides arepeptide fragments of FGFRs. They are derived from two distinct bindingsites for binding of the immunoglobulin-like module 2 of FGFR to NCAM F3modules 1-2. The invention further relates to use of said peptides forthe production of a medicament for the treatment of differentpathological conditions, wherein NCAM and/or FGFRs play a prominentrole.

BACKGROUND OF INVENTION

Fibroblast growth factor receptors (FGFRs) are a family of four closelyrelated receptor protein tyrosine kinases. Extracellularly they consistof three Ig-like modules and intracellularly of a split tyrosine-kinasemodule (Powers et al., 2000). The receptors are known as key regulatorsof morphogenesis, development, angiogenesis, and wound healing.

The fibroblast growth factor receptors (FGFR1-4) can be activated byvarious fibroblast growth factors (FGF1-23) (McKeehan et al., 1998; Itohand Ornitz, 2004) and cell adhesion molecules (CAMs) such as the neuralcell adhesion molecule (NCAM), CAM L1 and N-cadherin (Doherty and Walsh,1996; Kiselyov et al., 2003 and 2005). FGFR activation and signaling aredependent on dimerization of the receptor which is induced by a highaffinity binding of its ligand, fibroblast growth factor (FGF), and italso requires participation of cell surface heparin or heparan sulphateproteoglycans.

The extracellular part of the prototypical FGFR consists of three Igmodules of the intermediate subtype (Plotnikov et al., 1999; Pellegriniet al., 2000; Kiselyov et al., 2006a). The Ig2 and Ig3 modules mediatebinding to FGF and heparin, whereas the Ig1 module has anauto-inhibitory function (Wang et al., 1995; Olsen et al., 2004) bymeans of a direct binding to Ig2 (Kiselyov et al., 2006b).

NCAM is a cell surface glycoprotein belonging to the Ig superfamily ofCAMs (for review see Kiselyov et al., 2005). NCAM can be expressed asthree major isoforms (A, B and C) with differences in the cytoplasmicdomain. The extracellular part of NCAM is identical for the threeisoforms and consists of five Ig-like and two fibronectin type III (F3)modules. NCAM is widely expressed during embryonic development, whereasin the adult organism it is mainly found in tissues of neural origin.NCAM plays a major role during development of the nervous system,mediating adhesion between neural cells and stimulating neuriteoutgrowth and fasciculation, promoting cell survival and synapticplasticity (Cremer et al., 1997; Berezin et al., 2000; Bruses andRutishauser, 2001; Rougon and Hobert, 2003; Walmod et al., 2004). NCAMmediates cell-cell adhesion through homophilic binding and regulatesneurite outgrowth via FGFR (Doherty and Walsh, 1996; Kiselyov et al.,2003 and 2005). The FGFR site involved in binding to NCAM has beenmapped to the Ig3 module, and the corresponding site in NCAM—to thesecond F3 module (Kiselyov et al., 2003). The mechanism of the NCAMhomophilic binding, although extensively studied, is somewhatcontroversial, and for a thorough review of the available structuraldata, see Kiselyov et al., 2005. The Ig1 and Ig2 modules of NCAM weredemonstrated by surface plasmon resonance (SPR) to bind to each other,and it was suggested that these modules are involved in a symmetricaldouble reciprocal interaction (Kiselyov et al., 1997). These data werelater confirmed by several research groups using various methods such asnuclear magnetic resonance (NMR) analysis and X-ray crystallography(Jensen et al., 1999; Atkins et al., 1999; Kasper et al., 2000).Recently, the crystal structure of the first three N-terminal modules ofNCAM has been determined (Soroka et al., 2003), and based on thisstructure, a model of the NCAM homophilic binding has been suggested.According to this model, interaction between the Ig1 and Ig2 modulesleads to formation of a cis-dimer on the surface of the same cell. Thecis-dimers from two opposing cells can be involved in the formation oftwo kinds of one-dimensional “zippers”. When combined, the two “zippers”can form a two-dimensional “zipper”.

The different modules of NCAM have been shown to perform distinctfunctions. Thus, NCAM homophilic binding is believed to depend on thefirst three Ig modules. The heparin binding sequence is localized to theIg2 module. FGFR binding has been suggested to reside in the twomembrane-proximal F3 modules of NCAM.

The mechanism of FGFR activation by NCAM is not well understood. It isthought that most of the FGFR molecules are involved in a transientinteraction with NCAM (Kiselyov et al., 2003). When NCAM is not involvedin cell-cell adhesion, the NCAM molecules are supposed to be uniformlyspread on the cell surface. However, when NCAM is involved in cell-celladhesion (via the homophilic binding), NCAM molecules may arrangethemselves into the so-called ‘zipper’-formations (Soroka et al., 2003),which would lead to clustering of the NCAM molecules and, as a result,to clustering of the FGFR molecules. The increase in the localconcentration of the FGFR molecules is expected to increase the numberof the FGFR molecules involved in a direct FGFR-FGFR interaction, whichwould result in the increase of the background FGFR activation (Kiselyovet al., 2005).

It has previously been shown by SPR that the double-module construct ofNCAM F3 modules 1-2 binds to the double-module FGFR Ig2-1g3 constructwith a dissociation constant (Kd) of 10 μLM (Kiselyov et al., 2003).However, interaction between the individual modules could hardly bedetected by SPR. This indicates that both modules of the NCAM and FGFRconstructs are involved in the binding, or are necessary for thefull-strength binding. Using a more sensitive method (NMR), aninteraction between the NCAM F3 module 2 and FGFR Ig3 module has beendetected (the binding between the NCAM F3 module 1 and FGFR Ig2 modulewas not tested). The binding site in the NCAM F3 module 2 was mapped tothe FG-loop region of the module, which is located in the module'sN-terminus (Kiselyov et al., 2003). This indicates that the C-terminusof the NCAM F3 module 1 together with the N-terminal part of the NCAM F3module 2 could form a single binding site for FGFR, which may bedestroyed when the modules are separated.

SUMMARY OF INVENTION

The present invention describes two novel distinct NCAM binding sites ofFGFR Ig2 and discloses the use of peptide sequences derived from saidFGFR Ig2 binding sites capable of binding to NCAM and thereby modulatingNCAM signalling.

According to the invention a peptide sequence derived from the FGFR Ig2binding sites which is capable of binding to NCAM comprises about 25amino acid residues and comprises a fragment of FGFR Ig2.

The use according to the invention relates to induction ofdifferentiation, modulation of proliferation, stimulation ofregeneration, neuronal plasticity and survival of cells.

The invention relates to uses of the peptides for the treatment ofdifferent pathological conditions, wherein FGFR and/or NCAM plays a rolein pathology and/or recovery from disease, for example for

-   a) treatment of conditions of the central and peripheral nervous    system associated with postoperative nerve damage, traumatic nerve    damage, impaired myelination of nerve fibers, postischaemic damage,    e.g. resulting from a stroke, Parkinson's disease, Alzheimer's    disease, Huntington's disease, dementias such as multiinfarct    dementia, sclerosis, nerve degeneration associated with diabetes    mellitus, disorders affecting the circadian clock or neuromuscular    transmission, and schizophrenia, mood disorders, such as manic    depression;-   b) treatment of diseases or conditions of the muscles including    conditions with impaired function of neuromuscular connections, such    as after organ transplantation, or such as genetic or traumatic    atrophic muscle disorders; or for treatment of diseases or    conditions of various organs, such as degenerative conditions of the    gonads, of the pancreas such as diabetes mellitus type I and II, of    the kidney such as nephrosis and of the heart, liver and bowel;-   c) promotion of wound-healing;-   d) prevention of death of heart muscle cells, such as after acute    myocardial infarction;-   e) promotion of revascularisation;-   f) stimulation of the ability to learn and/or the short and/or    long-term memory;-   g) treatment of cancer.

DESCRIPTION OF DRAWINGS

FIG. 1 shows SPR analysis of the binding between the FGFR Ig2 module andNCAM F3 modules 1-2

-   A) Binding of soluble FGFR Ig2 module to the immobilized NCAM F3    modules.-   B) Binding of soluble NCAM F3 modules to the immobilized FGFR Ig2    module. The experiment was repeated 9 times.

FIG. 2 shows mapping of the Ig2 module's residues involved in binding toNCAM. Changes in chemical shifts of 10 μM ¹⁵N labelled Ig1 module afteraddition of 13 (A), 25 (B) and 40 (C) μM unlabeled NCAM F3 modules 1-2and mapping of the significantly perturbed residues onto the structureof the FGFR 1g2 module (right panels). The change of the chemical shiftwas calculated using the following expression: ((5*ΔH)̂2+(ΔN)̂2)″0.5,where ΔH is the change of the ¹H chemical shift and ΔN is the change ofthe ¹⁵N chemical shift.

FIG. 3 shows mapping of the various binding sites of the FGFR 1g2 moduleonto the module's structure.

FIG. 4 demonstrates SPR analysis of the inhibitory effect of sucroseoctasulphate (SOS) on the FGFR Ig2-NCAM F3(1-2) binding. The binding of20 μM 1g2 module of FGFR to the immobilized NCAM F3 modules 1-2 wasperformed using indicated concentrations of SOS. The experiment wasrepeated 9 times.

FIG. 5 demonstrates binding of the FGFR1-Ig2 derived peptide AKTVKFK(amino acids 171-177) (SEQ ID NO:2) to the immobilized fibronectinmodules (F3(1-2)) of NCAM.

FIG. 6 demonstrates binding of the FGFR1-Ig2 derived peptide RWLKNGKEFK(amino acids 189-198) (SEQ ID NO:3) to the immobilized fibronectinmodules (F3(1-2)) of NCAM.

DETAILED DESCRIPTION OF THE INVENTION

A peptide for use according to the invention can be derived from anyFGFR, such as FGFR1, FGFR2, FGFR3, FGFR4 and FGFR5 or it may be derivedfrom a variant of any of FGFR1-5, such as a natural or recombinant FGFRvariant, for example a FGFR variant produced by alternative splicing,e.g. FGFR1b or FGFR2b. or genetic polymorphism, or any type ofrecombinant FGFR. It is to be understood that a FGFR of the inventionand a variant thereof comprise the NCAM F3 moduleI-2 binding sitesdescribed herein, or comprise at least a part of said binding sites.Examples of FGFRs of the invention which comprise the NCAM F3 module-2binding sites of the invention may be the FGFR polypeptides identifiedin the GenBank database as Ass. Nos: P11362 (corresponding to humanFGFR1), P16092 (corresponding to mouse FGFR1), P21802 (corresponding tohuman FGFR2), P21803 (corresponding to mouse FGFR2) P22607(corresponding to human FGFR3), Q61861 (corresponding to mouse FGFR3)

P22455 (corresponding to human FGFR4), Q03142 (corresponding to mouseFGFR4) or AAK26742 (corresponding to human FGFR5).

The peptide for use according to the invention is a peptide which iscapable of modulating activity of NCAM. In one embodiment the peptidemay be capable of activating NCAM. In another embodiment, the peptidemay be capable of inhibiting NCAM. By the terms “modulation” or“modulating” are meant a change, such as an inhibition or stimulation.

1. Amino Acid Sequence

Peptides for use according to the invention comprise a fragment of FGFRIg2 which comprises a contiguous amino acid sequence derived from twodistinct NCAM F3 module 1-2 binding sites or a fragment, homologue orvariant thereof. The first binding site comprises a cluster involvingresidues T¹⁵⁶, S¹⁵⁷, E¹⁵⁹, A¹⁷¹, T¹⁷³, V¹⁷⁴ _(v)K¹⁷⁵, S¹⁸¹, S²¹⁴, M²¹⁷,D²¹⁸, S²¹⁹ and V²²⁰ from the region a.a 140-251 of FGFR corresponding toFGFR Ig2, and the second binding site comprises a cluster involvingresidues M¹⁶¹, L¹⁹¹, K¹⁹², N¹⁹³, F¹⁹⁷, V²²¹, T²²⁹, C²³⁰, D²⁴⁶ and V²⁴⁸,from the region a.a 140-251 of FGFR corresponding to FGFR Ig2.

In a preferred embodiment the peptides for use according to theinvention may comprise a contiguous amino acid sequence which is derivedfrom FGFR Ig2. Accordingly, in this embodiment the amino acid sequencefor use according to the invention may be selected from the followingamino acid sequences:

(SEQ ID NO: 1) TSPEKMEKKL (SEQ ID NO: 2) AKTVKFK (SEQ ID NO: 3)RWLKNGKEFK (SEQ ID NO: 4) TWSIIMDSV (SEQ ID NO: 5) SDKGNYTCIVEN(SEQ ID NO: 6) TYQLDVVERS,or a fragment, variant or homologue thereof.

In one embodiment a homologue for use according to the inventioncomprises the motif X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-x13 andfragments or variants thereof, wherein

X1 is T or no amino acid,X3 is a charged amino acid,

X4 is W, X6 is K,

X5, X7 and X8 are any amino acid,X2, X9, X10, X11, X12, and X13 are any or no amino acid,X9 is any or no amino acid,X10 is any or no amino acid,X11 is any or no amino acid,X12 is any or no amino acid,X13 is any or no amino acid.

Examples of homologues may be:

(SEQ ID NO: 7) TIRWLKNG (CNTN1, SwissProt ID: Q12860) (SEQ ID NO: 8)KWLKNGKE (pro-neuroregulin, SwissProt ID: Q05199) (SEQ ID NO: 9)TLRWFKNGQ (neurofascin, SwissProt ID: Q9QVN5, 094856) (SEQ ID NO: 10)TIRWFKGNKELK (nectin-like-2, SwissProt ID: Q1WIL9, Q6AYP5)(SEQ ID NO: 11) IRWFKNDKEIK (nectin-like-3, SwissProt ID: Q1WIM2)(SEQ ID NO: 12) RWTKDGIHFKP (L1, SwissProt ID: Q9QyQ7). (SEQ ID NO: 13)TYRWLKNGVPLSP (CNTN5, SwissProt ID: Q49AF3).

In another embodiment a homologue for use according to the inventioncomprises the motif X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12 and fragmentsor variants thereof, wherein

X1 is S, X2 is D, X4 is G, X6 is Y, X8 is C,

X3, X5, X7, X9, X10, X11, and X12 are any amino acid.

Examples of homologues may be:

(SEQ ID NO: 14) SDVGNYTCVVTN (CNTN4, SwissProt ID: Q14BL8, Q8IWV2)(SEQ ID NO: 15) SDVGNYTCFVTN (CNTN6, SwissProt ID: P97528)(SEQ ID NO: 16) SDEGKYTCFAEN (CNTN2, SwissProt ID: Q02246)(SEQ ID NO: 17) SDKGNYSCFVSS (CNTN1, SwissProt ID: Q12860, Q28106)(SEQ ID NO: 18) SDSGNYTCMAAN (Netrin receptor UNC5D precursor,SwissProt ID: Q8K1S2)

In the present context the standard one-letter code for amino acidresidues as well as the standard three-letter code are applied.Abbreviations for amino acids are in accordance with the recommendationsin the IUPAC-IUB Joint Commission on Biochemical Nomenclature Eur. J.Biochem, 1984, vol. 184, pp 9-37. Throughout the description and claimseither the three letter code or the one letter code for natural aminoacids are used. Where the L or D form has not been specified it is to beunderstood that the amino acid in question has the natural L form, cf.Pure & Appl. Chem. Vol. (56(5) pp 595-624 (1984) or the D form, so thatthe peptides formed may be constituted of amino acids of L form, D form,or a sequence of mixed L forms and D forms.

Where nothing is specified it is to be understood that the C-terminalamino acid of a peptide for use according to the invention exists as thefree carboxylic acid, this may also be specified as “—OH”. However, theC-terminal amino acid of a peptide for use according to the inventionmay be the amidated derivative, which is indicated as “—NH2”. Wherenothing else is stated the N-terminal amino acid of a polypeptidecomprises a free amino-group, this may also be specified as “H—”.

A peptide, fragment, homologue or variant for use according to theinvention can also comprise one or several unnatural amino acids.

A preferred peptide for use according to the invention is an isolatedcontiguous peptide sequence which comprises at most 25 amino acidresidues. In one embodiment the length of the amino acid sequence of apeptide may be from 3 to 10 amino acid residues, such as for example 4,5, 6, 7, 8, or 9 amino acid residues. In another embodiment, the lengthof the amino acid sequence of a peptide may be from 11-25 amino acidresidues, such as for example 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23 or 24 amino acid residues. The peptides which amino acid sequencehas the length in the range of 5 to 15 amino acid residues, such as from6 to 13, for example 7, 8, 9, 10, 11, or 12, are preferred. It isunderstood that all peptides for use according to the invention compriseat least one amino acid sequence selected from any of the sequences SEQID NOs: 1-6 or a fragment, variant or homologue thereof.

Thus, some embodiments of the invention may relate to the use of apeptide comprising a fragment of a sequence selected from SEQ IDNOs:1-6. Another embodiment may relate to the use of variants of SEQ IDNOs:1-6. A further embodiment may relate to the use of homologues of SEQID NOs: 1-6.

For use according to the invention, a variant of an amino acid sequenceselected from the sequences SEQ ID NOs: 1-6 may be

-   -   i) an amino acid sequence which has at least 75% identity with a        selected sequence, such as 76-80% identity, for example 81-85%        identity, such as 86-90% identity, for example 91-95% identity,        such as 96-99% identity, wherein the identity is defined as a        percentage of identical amino acids in said sequence when it is        collated with the selected sequence. The identity between amino        acid sequences may be calculated using well known algorithms        such as BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55,        BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM        80, BLOSUM 85, or BLOSUM 9;    -   ii) an amino acid sequence which has at least 75% positive amino        acid matches with a selected sequence, such as 76-80% positive        amino acid matches, for example 81-85% positive amino acid        matches, such as 86-90% positive amino acid matches, for example        91-95% positive amino acid matches, such as 96-99% positive        amino acid matches, wherein the positive amino acid match is        defined as the presence at the same position in two compared        sequences of amino acid residues which has similar physical        and/or chemical properties. Preferred positive amino acid        matches of the present invention are K to R, E to D, L to M, Q        to E, I to V, I to L, A to S, Y to W, K to Q, S to T, N to S and        Q to R;    -   iii) an amino acid sequence which is identical to a selected        sequence, or it has at least 75% identity with said sequence        such as 76-80% identity, for example 81-85% identity, such as        86-90% identity, for example 91-95% identity, such as 96-99%        identity, or has at least 75% positive amino acid matches with        the selected sequence, such as 76-80% positive amino acid        matches, for example 81-85% positive amino acid matches, such as        86-90% positive amino acid matches, for example 91-95% positive        amino acid matches, such as 96-99% positive amino acid matches,        and comprises other chemical moieties, e.g. phosphoryl, sulphur,        acetyl, glycosyl moieties.

The term “variant of a peptide sequence” also means that the peptidesequence may be modified, for example by substitution of one or more ofthe amino acid residues. Both L-amino acids and D-amino acids may beused. Other modification may comprise derivatives such as esters,sugars, etc., for example methyl and acetyl esters.

In another aspect, variants of the amino acid sequences used accordingto the invention may comprise, within the same variant, or fragmentsthereof or among different variants, or fragments thereof, at least onesubstitution, such as a plurality of substitutions introducedindependently of one another. Variants of the complex, or fragmentsthereof may thus comprise conservative substitutions independently ofone another, wherein at least one glycine (Gly) of said variant, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Ala, Val, Leu, and Ile, andindependently thereof, variants, or fragments thereof, wherein at leastone alanine (Ala) of said variants, or fragments thereof is substitutedwith an amino acid selected from the group of amino acids consisting ofGly, Val, Leu, and Ile, and independently thereof, variants, orfragments thereof, wherein at least one valine (Val) of said variant, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Gly, Ala, Leu, and Ile, andindependently thereof, variants, or fragments thereof, wherein at leastone leucine (Leu) of said variant, or fragments thereof is substitutedwith an amino acid selected from the group of amino acids consisting ofGly, Ala, Val, and Ile, and independently thereof, variants, orfragments thereof, wherein at least one isoleucine (Ile) of saidvariants, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Gly, Ala, Val andLeu, and independently thereof, variants, or fragments thereof whereinat least one aspartic acids (Asp) of said variant, or fragments thereofis substituted with an amino acid selected from the group of amino acidsconsisting of Glu, Asn, and Gln, and independently thereof, variants, orfragments thereof, wherein at least one aspargine (Asn) of saidvariants, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Asp, Glu, and Gln,and independently thereof, variants, or fragments thereof, wherein atleast one glutamine (Gin) of said variants, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Asp, Glu, and Asn, and wherein at least one phenylalanine(Phe) of said variants, or fragments thereof is substituted with anamino acid selected from the group of amino acids consisting of Tyr,Trp, His, Pro, and preferably selected from the group of amino acidsconsisting of Tyr and Trp, and independently thereof, variants, orfragments thereof, wherein at least one tyrosine (Tyr) of said variants,or fragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Phe, Trp, His, Pro, preferably anamino acid selected from the group of amino acids consisting of Phe andTrp, and independently thereof, variants, or fragments thereof, whereinat least one arginine (Arg) of said fragment is substituted with anamino acid selected from the group of amino acids consisting of Lys andHis, and independently thereof, variants, or fragments thereof, whereinat least one lysine (Lys) of said variants, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Arg and His, and independently thereof, variants, orfragments thereof, and independently thereof, variants, or fragmentsthereof, and wherein at least one proline (Pro) of said variants, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Phe, Tyr, Trp, and His, andindependently thereof, variants, or fragments thereof, wherein at leastone cysteine (Cys) of said variants, or fragments thereof is substitutedwith an amino acid selected from the group of amino acids consisting ofAsp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr, and Tyr.

It thus follows from the above that the same functional equivalent of apeptide fragment, or fragment of said functional equivalent may comprisemore than one conservative amino acid substitution from more than onegroup of conservative amino acids as defined herein above. The term“conservative amino acid substitution” is used synonymously herein withthe term “homologous amino acid substitution”.

The groups of conservative amino acids are as the following: A, G

(neutral, weakly hydrophobic),Q, N, S, T (hydrophilic, non-charged)E, D (hydrophilic, acidic)H, K, R (hydrophilic, basic)L, P, I, V, M, F, Y, W (hydrophobic, aromatic)C (cross-link forming)

Conservative substitutions may be introduced in any position of apreferred predetermined peptide for use according to the invention orfragment thereof. It may however also be desirable to introducenon-conservative substitutions, particularly, but not limited to, anon-conservative substitution in any one or more positions.

A non-conservative substitution leading to the formation of afunctionally equivalent fragment of the peptide for use according to theinvention would for example differ substantially in polarity, forexample a residue with a non-polar side chain (Ala, Leu, Pro, Trp, Val,Ile, Leu, Phe or Met) substituted for a residue with a polar side chainsuch as Gly, Ser, Thr, Cys, Tyr, Asn, or Gln or a charged amino acidsuch as Asp, Glu, Arg, or Lys, or substituting a charged or a polarresidue for a non-polar one; and/or ii) differ substantially in itseffect on peptide backbone orientation such as substitution of or forPro or Gly by another residue; and/or iii) differ substantially inelectric charge, for example substitution of a negatively chargedresidue such as Glu or Asp for a positively charged residue such as Lys,His or Arg (and vice versa); and/or iv) differ substantially in stericbulk, for example substitution of a bulky residue such as His, Trp, Pheor Tyr for one having a minor side chain, e.g. Ala, Gly or Ser (and viceversa).

Substitution of amino acids may in one embodiment be made based upontheir hydrophobicity and hydrophilicity values and the relativesimilarity of the amino acid side-chain substituents, including charge,size, and the like.

Both fragments and variants of amino acid sequences for use according tothe invention are the functional equivalents of said sequences.

By the term “functional equivalent” of an amino acid sequence is in thepresent context meant a molecule which meets the criteria for a variantor a fragment of said amino acid sequence described above and which iscapable of one or more functional activities of said sequence or acompound comprising said sequence. In a preferred embodiment thefunctional equivalent of an amino acid sequence for use according to theinvention is capable of binding and modulating activity of NCAM.

The invention relates both to isolated peptides for use according to theinvention and fusion proteins comprising peptides for use according tothe invention.

In one embodiment, the peptide for use according to the invention is anisolated peptide. By the term “isolated peptide” is meant that thepeptide for use according to the invention is an individual compound andnot a part of another compound, such as for example a polypeptidecomprising more than 25 amino acid residues. The isolated peptide may beproduced by use of any recombinant technology methods or chemicalsynthesis and separated from other compounds, or it may be separatedfrom a longer polypeptide or protein by a method of enzymatic orchemical cleavage and further separated from other protein fragments.

An isolated peptide for use according to the invention may in oneembodiment comprise a fragment of FGFR Ig2 which comprises a contiguousamino acid sequence derived from two distinct NCAM F3 module 1-2 bindingsites. The peptide with Seq ID NO:1 contains three a.a from the firstbinding site and one a.a. from the second binding site. The peptide withSeq ID NO:2 contains four a.a. from the first binding site. The peptidewith Seq. ID NO:3 contains four a.a from the second binding site. Thepeptide with Seq ID NO:4 contains four a.a from the first binding site.The peptide with Seq ID NO:5 contains two a.a. from the second bindingsite. The peptide with Seq ID NO:6 contains two a.a from the secondbinding site. An isolated peptide for use according to the invention mayin one embodiment comprise a fragment of FGFR Ig2 which comprises acontiguous amino acid sequence derived from two distinct NCAM F3 module1-2 binding sites, selected from SEQ ID NOs:1-6 or a fragment orhomologue thereof. In another embodiment the isolated peptide mayconsist of one or more of the sequences SEQ ID Nos:1-6.

Production of Peptide Sequences

The peptide sequences of the present invention may be prepared by anyconventional synthetic methods, recombinant DNA technologies, enzymaticcleavage of full-length proteins which the peptide sequences are derivedfrom, or a combination of said methods.

Recombinant Preparation

Thus, in one embodiment the peptides of the invention are produced byuse of recombinant DNA technologies.

The DNA sequence encoding a peptide or the corresponding full-lengthprotein the peptide originates from may be prepared synthetically byestablished standard methods, e.g. the phosphoamidine method describedby Beaucage and Caruthers, 1981, Tetrahedron Lett. 22:1859-1869, or themethod described by Matthes et al., 1984, EMBO J. 3:801-805. Accordingto the phosphoamidine method, oligonucleotides are synthesised, e.g. inan automatic DNA synthesiser, purified, annealed, ligated and cloned insuitable vectors.

The DNA sequence encoding a peptide may also be prepared byfragmentation of the DNA sequences encoding the correspondingfull-length protein of peptide origin, using DNAase I according to astandard protocol (Sambrook et al., Molecular cloning: A Laboratorymanual. 2 rd ed., CSHL Press, Cold Spring Harbor, N.Y., 1989). Thepresent invention relates to full-length proteins selected from thegroups of proteins identified above. The DNA encoding the full-lengthproteins of the invention may alternatively be fragmented using specificrestriction endonucleases. The fragments of DNA are further purifiedusing standard procedures described in Sambrook et al., Molecularcloning: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor,N.Y., 1989.

The DNA sequence encoding a full-length protein may also be of genomicor cDNA origin, for instance obtained by preparing a genomic or cDNAlibrary and screening for DNA sequences coding for all or part of thefull-length protein by hybridisation using synthetic oligonucleotideprobes in accordance with standard techniques (cf. Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989). The DNA sequence may also be prepared by polymerase chainreaction using specific primers, for instance as described in U.S. Pat.No. 4,683,202 or Saiki et al., 1988, Science 239:487-491.

The DNA sequence is then inserted into a recombinant expression vector,which may be any vector, which may conveniently be subjected torecombinant DNA procedures. The choice of vector will often depend onthe host cell into which it is to be introduced. Thus, the vector may bean autonomously replicating vector, i.e. a vector that exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated.

In the vector, the DNA sequence encoding a peptide or a full-lengthprotein should be operably connected to a suitable promoter sequence.The promoter may be any DNA sequence, which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or heterologous to the host cell.Examples of suitable promoters for directing the transcription of thecoding DNA sequence in mammalian cells are the SV 40 promoter (Subramaniet al., 1981, Mol. Cell Biol. 1:854-864), the MT-1 (metallothioneingene) promoter (Palmiter et al., 1983, Science 222: 809-814) or theadenovirus 2 major late promoter. A suitable promoter for use in insectcells is the polyhedrin promoter (Vasuvedan et al., 1992, FEBS Lett.311:7-11). Suitable promoters for use in yeast host cells includepromoters from yeast glycolytic genes (Hitzeman et al., 1980, J. Biol.Chem. 255:12073-12080; Alber and Kawasaki, 1982, J. Mol. Appl. Gen. 1:419-434) or alcohol dehydrogenase genes (Young et al., 1982, in GeneticEngineering of Microorganisms for Chemicals, Hollaender et al, eds.,Plenum Press, New York), or the TPI1 (U.S. Pat. No. 4,599,311) orADH2-4-c (Russell et al., 1983, Nature 304:652-654) promoters. Suitablepromoters for use in filamentous fungus host cells are, for instance,the ADH3 promoter (McKnight et al., 1985, EMBO J. 4:2093-2099) or thetpiA promoter.

The coding DNA sequence may also be operably connected to a suitableterminator, such as the human growth hormone terminator (Palmiter etal., op. cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki, op.cit.) or ADH3 (McKnight et al., op. cit.) promoters. The vector mayfurther comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 Elb region), transcriptional enhancer sequences(e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. theones encoding adenovirus VA RNAs).

The recombinant expression vector may further comprise a DNA sequenceenabling the vector to replicate in the host cell in question. Anexample of such a sequence (when the host cell is a mammalian cell) isthe SV 40 origin of replication. The vector may also comprise aselectable marker, e.g. a gene the product of which complements a defectin the host cell, such as the gene coding for dihydrofolate reductase(DHFR) or one which confers resistance to a drug, e.g. neomycin,hydromycin or methotrexate.

The procedures used to ligate the DNA sequences coding the peptides orfull-length proteins, the promoter and the terminator, respectively, andto insert them into suitable vectors containing the informationnecessary for replication, are well known to persons skilled in the art(cf., for instance, Sambrook et al., op.cit.).

To obtain recombinant peptides of the invention the coding DNA sequencesmay be usefully fused with a second peptide coding sequence and aprotease cleavage site coding sequence, giving a DNA construct encodingthe fusion protein, wherein the protease cleavage site coding sequencepositioned between the HBP fragment and second peptide coding DNA,inserted into a recombinant expression vector, and expressed inrecombinant host cells. In one embodiment, said second peptide selectedfrom, but not limited by the group comprising glutathion-5-reductase,calf thymosin, bacterial thioredoxin or human ubiquitin natural orsynthetic variants, or peptides thereof. In another embodiment, apeptide sequence comprising a protease cleavage site may be the FactorXa, with the amino acid sequence IEGR, enterokinase, with the amino acidsequence DDDDK, thrombin, with the amino acid sequence LVPR/GS, orAcharombacter lyticus, with the amino acid sequence XKX, cleavage site.

The host cell into which the expression vector is introduced may be anycell which is capable of expression of the peptides or full-lengthproteins, and is preferably a eukaryotic cell, such as invertebrate(insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes ormammalian cells, in particular insect and mammalian cells. Examples ofsuitable mammalian cell lines are the HEK293 (ATCC CRL-1573), COS (ATCCCRL-1650), BHK (ATCC CRL-1632, ATCC CCL-10) or CHO (ATCC CCL-61) celllines. Methods of transfecting mammalian cells and expressing DNAsequences introduced in the cells are described in e.g. Kaufman andSharp, J. Mol. Biol. 159, 1982, pp. 601621; Southern and Berg, 1982, J.Mol. Appl. Genet. 1:327-341; Loyter et al., 1982, Proc. Natl. Acad. Sci.USA 79: 422-426; Wigler et al., 1978, Cell 14:725; Corsaro and Pearson,1981, in Somatic Cell Genetics 7, p. 603; Graham and van der Eb, 1973,Virol. 52:456; and Neumann et al., 1982, EMBO J. 1:841-845.

Alternatively, fungal cells (including yeast cells) may be used as hostcells. Examples of suitable yeast cells include cells of Saccharomycesspp. or Schizosaccharomyces spp., in particular strains of Saccharomycescerevisiae. Examples of other fungal cells are cells of filamentousfungi, e.g. Aspergillus spp. or Neurospora spp., in particular strainsof Aspergillus oryzae or Aspergillus niger. The use of Aspergillus spp.for the expression of proteins is described in, e.g., EP 238 023.

The medium used to culture the cells may be any conventional mediumsuitable for growing mammalian cells, such as a serum-containing orserum-free medium containing appropriate supplements, or a suitablemedium for growing insect, yeast or fungal cells. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g. in catalogues of the American Type CultureCollection).

The peptides or full-length proteins recombinantly produced by the cellsmay then be recovered from the culture medium by conventional proceduresincluding separating the host cells from the medium by centrifugation orfiltration, precipitating the proteinaceous components of thesupernatant or filtrate by means of a salt, e.g. ammonium sulphate,purification by a variety of chromatographic procedures, e.g. HPLC, ionexchange chromatography, affinity chromatography, or the like.

Synthetic Preparation

The methods for synthetic production of peptides are well known in theart. Detailed descriptions as well as practical advice for producingsynthetic peptides may be found in Synthetic Peptides: A User's Guide(Advances in Molecular Biology), Grant G. A. ed., Oxford UniversityPress, 2002, or in: Pharmaceutical Formulation: Development of Peptidesand Proteins, Frokjaer and Hovgaard eds., Taylor and Francis, 1999.

Peptides may for example be synthesised by using Fmoc chemistry and withAcm-protected cysteins. After purification by reversed phase HPLC,peptides may be further processed to obtain for example cyclic or C- orN-terminal modified isoforms. The methods for cyclization and terminalmodification are well-known in the art and described in detail in theabove-cited manuals.

In a preferred embodiment the peptide sequences of the invention areproduced synthetically, in particular, by the Sequence Assisted PeptideSynthesis (SAPS) method.

Peptides may be synthesised either batchwise in a polyethylene vesselequipped with a polypropylene filter for filtration or in thecontinuous-flow version of the polyamide solid-phase method (Dryland, A.and Sheppard, R C., (1986) J. Chem. Soc. Perkin Trans. I, 125-137) on afully automated peptide synthesiser using 9-fluorenylmethyloxycarbonyl(Fmoc) or tert.-Butyloxycarbonyl, (Boc) as N-a-amino protecting groupand suitable common protection groups for side-chain functionality's.

Medicament

It is an objective of the invention to provide a compound capable ofmodulating the activity of NCAM, said compound being concerned by theinvention as a medicament for the treatment of diseases, whereinmodulation of NCAM signalling may be considered as an essentialcondition for curing.

Accordingly, the invention relates to the use of one or more of thepeptides comprising a sequence corresponding to a Ig2 NCAM binding siteof the FGF receptor or a fragment thereof or a variant for themanufacture of a medicament.

In one embodiment the medicament of the invention comprises at least oneof the amino acid sequences set forth in SEQ ID NOS: 1-6 or fragments orvariants or homologues of said sequences, or fragments or variants ofsaid homologues. In another embodiment the medicament of the inventioncomprises an antibody capable of binding to an epitope comprising abinding site of the invention or a fragment or variant of said antibody.

The medicament may in one aspect prevent death of cells in vitro or invivo, wherein the composition is administered to a subject, in vitro orin vivo in an effective amount of one or more of the compounds describedabove or a composition as described below, so as to prevent cell deathof NCAM presenting cells in several tissues and organs as discussedherein.

The medicament of the invention comprises an effective amount of one ormore of the compounds as defined above, or a composition comprisingcompound as defined above, in combination with pharmaceuticallyacceptable additives. Such medicament may suitably be formulated fororal, percutaneous, intramuscular, intravenous, intracranial,intrathecal, intracerebroventricular, intranasal or pulmonaladministration.

Strategies in formulation development of medicaments and compositionsbased on the peptides of the present invention generally correspond toformulation strategies for any other protein-based drug product.Potential problems and the guidance required to overcome these problemsare dealt with in several textbooks, e.g. “Therapeutic Peptides andProtein Formulation. Processing and Delivery Systems”, Ed. A. K. Banga,Technomic Publishing AG, Basel, 1995.

Injectables are usually prepared either as liquid solutions orsuspensions, solid forms suitable for solution in, or suspension in,liquid prior to injection. The preparation may also be emulsified. Theactive ingredient is often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol or the like, and combinations thereof. In addition, if desired,the preparation may contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, pH buffering agents, or which enhancethe effectiveness or transportation of the preparation.

Formulations of the compounds of the invention can be prepared bytechniques known to the person skilled in the art. The formulations maycontain pharmaceutically acceptable carriers and excipients includingmicrospheres, liposomes, microcapsules, nanoparticles or the like.

The preparation may suitably be administered by injection, optionally atthe site, where the active ingredient is to exert its effect. Additionalformulations which are suitable for other modes of administrationinclude suppositories, nasal, pulmonal and, in some cases, oralformulations. For suppositories, traditional binders and carriersinclude

polyalkylene glycols or triglycerides. Such suppositories may be formedfrom mixtures containing the active ingredient(s) in the range of from0.5% to 10%, preferably 1-2%. Oral formulations include such normallyemployed excipients as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, and the like. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and generally contain 10-95% of the activeingredient(s), preferably 25-70%.

Other formulations are such suitable for nasal and pulmonaryadministration, e.g. inhalators and aerosols.

The active compound may be formulated as neutral or salt forms.Pharmaceutically acceptable salts include acid addition salts (formedwith the free amino groups of the peptide compound) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic acid, oxalic acid, tartaric acid,mandelic acid, and the like. Salts formed with the free carboxyl groupmay also be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The preparations are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective.The quantity to be administered depends on the subject to be treated,including, e.g. the weight and age of the subject, the disease to betreated and the stage of disease. Suitable dosage ranges are per kilobody weight normally of the order of several hundred μg activeingredient per administration with a preferred range of from about 0.1μg to 5000 μg per kilo body weight. Using monomeric forms of thecompounds, the suitable dosages are often in the range of from 0.1 μg to5000 μg per kilo body weight, such as in the range of from about 0.1 μgto 3000 μg per kilo body weight, and especially in the range of fromabout 0.1 pg to 1000 μg per kilo body weight. Using multimeric forms ofthe compounds, the suitable dosages are often in the range of from 0.1μg to 1000 μg per kilo body weight, such as in the range of from about0.1 vg to 750 μg per kilo body weight, and especially in the range offrom about 0.1 μg to 500 μg per kilo body weight such as in the range offrom about 0.1 μg to 250 μg per kilo body weight. In particular whenadministering nasally smaller dosages are used than when administeringby other routes. Administration may be performed once or may be followedby subsequent administrations. The dosage will also depend on the routeof administration and will vary with the age and weight of the subjectto be treated. A preferred dosage of multimeric forms would be in theinterval 1 mg to 70 mg per 70 kg body weight.

For most indications a localised or substantially localised applicationis preferred.

Some of the compounds of the present invention are sufficiently active,but for some of the others, the effect will be enhanced if thepreparation further comprises pharmaceutically acceptable additivesand/or carriers. Such additives and carriers will be known in the art.In some cases, it will be advantageous to include a compound, whichpromote delivery of the active substance to its target.

In many instances, it will be necessary to administrate the formulationmultiple times. Administration may be a continuous infusion, such asintraventricular infusion or administration in more doses such as moretimes a day, daily, more times a week, weekly, etc. It is preferred thatadministration of the medicament is initiated before or shortly afterthe individual has been subjected to the factor(s) that may lead to celldeath. Preferably the medicament is administered within 8 hours from thefactor onset, such as within 5 hours from the factor onset. Many of thecompounds exhibit a long term effect whereby administration of thecompounds may be conducted with long intervals, such as 1 week or 2weeks.

In connection with the use in nerve guides, the administration may becontinuous or in small portions based upon controlled release of theactive compound(s). Furthermore, precursors may be used to control therate of release and/or site of release. Other kinds of implants and wellas oral administration may similarly be based upon controlled releaseand/or the use of precursors.

As discussed above, the present invention relates to treatment ofindividuals for inducing differentiation, modulating proliferation,stimulate regeneration, neuronal plasticity and survival of NCAMpresenting cells in vitro or in vivo, the treatment involvingadministering an effective amount of one or more compounds as definedabove.

Another strategy for administration is to implant or inject cellscapable of expressing and secreting the compound in question. Therebythe compound may be produced at the location where it is going to act.

Treatment

Treatment according to the invention is in one embodiment useful forinducing differentiation, modulating proliferation, stimulatingregeneration, neuronal plasticity and survival of cells, for examplecells being implanted or transplanted.

In further embodiment the treatment may be for stimulation of survivalof cells which are at risk of dying due to a variety of factors, such astraumas and injuries, acute diseases, chronic diseases and/or disorders,in particular degenerative diseases normally leading to cell death,other external factors, such as medical and/or surgical treatmentsand/or diagnostic methods that may cause formation of free radicals orotherwise have cytotoxic effects, such as X-rays and chemotherapy. Inrelation to chemotherapy the NCAM binding peptides used according to theinvention are useful in cancer treatment.

Thus, the treatment comprises treatment and/or prophylaxis of cell deathin relation to diseases or conditions of the central and peripheralnervous system, such as postoperative nerve damage, traumatic nervedamage, e.g. resulting from spinal cord injury, impaired myelination ofnerve fibers, postischaemic damage, e.g. resulting from a stroke,multiinfarct dementia, multiple sclerosis, nerve degeneration associatedwith diabetes mellitus, neuromuscular degeneration, schizophrenia,Alzheimer's disease, Parkinson's disease, or Huntington's disease.

Also, in relation to diseases or conditions of the muscles includingconditions with impaired function of neuromuscular connections, such asgenetic or traumatic atrophic muscle disorders; or for the treatment ofdiseases or conditions of various organs, such as degenerativeconditions of the gonads, of the pancreas, such as diabetes mellitustype I and II, of the kidney, such as nephrosis the compounds accordingto the invention may be used for inducing differentiation, modulatingproliferation, stimulate regeneration, neuronal plasticity and survival,i.e. stimulating survival.

Furthermore, the treatment may be for preventing cell death of heartmuscle cells, such as after acute myocardial infarction, in order toinduce angiogenesis. Furthermore, in one embodiment the treatment is forthe stimulation of the survival of heart muscle cells, such as survivalafter acute myocardial infarction. In another aspect the treatment isfor revascularisation, such as after injuries.

It is also within the scope of the invention a use of the peptides forthe promotion of wound-healing. The present peptides are capable ofstimulating angiogenesis and thereby they can promote the wound healingprocess.

The invention further discloses a use of peptides in the treatment ofcancer. Regulation of activation of NCAM is important for tumorangiogenesis, proliferation and spreading.

In yet a further embodiment a use of the peptides is for the stimulationof the ability to learn and/or of the short and/or long term memory, asNCAM activity is important for differentiation of neural cells.

In still another embodiment a peptide for use according to the inventionis for the treatment of body damages due to alcohol consumption.Developmental malformations of foetuses, long-term neurobehavioralalterations, alcoholic liver disease are particularly concerned.

Therapeutic treatment of prion diseases including using a peptide isstill another embodiment of the invention.

In particular the use according to the invention of a peptide may be forthe treatment of clinical conditions, such as neoplasms such asmalignant neoplasms, benign neoplasms, carcinoma in situ and neoplasmsof uncertain behavior, cancer in breast, thyroidal, pancreas, brain,lung, kidney, prostate, liver, heart, skin, blood organ, muscles(sarcoma), cancers with dysfunction and/or over- or under-expression ofspecific receptors and/or expression of mutated receptors or associatedwith soluble receptors, such as but not limited to Erb-receptors andFGF-receptors, diseases of endocrine glands, such as diabetes mellitus Iand II, pituitary gland tumor, psychoses, such as senile and presenileorganic psychotic conditions, alcoholic psychoses, drug psychoses,transient organic psychotic conditions, Alzheimer's disease, cerebrallipidoses, epilepsy, general paresis [syphilis], hepatolenticulardegeneration, Huntington's chorea, Jakob-Creutzfeldt disease, multiplesclerosis, Pick's disease of the brain, polyareriti nodosa, syphilis,schizophrenic disorders, affective psychoses, neurotic disorders,personality disorders, including character neurosis, nonpsychoticpersonality disorder associated with organic brain syndromes, paranoidpersonality disorder, fanatic personality, paranoid personality(disorder), paranoid traits, sexual deviations and disorders ordysfunctions (including reduced sexual drive for whatever reason),mental retardation, disease in the nerve system and sense organs, suchas affecting sight, hearing, smell, feeling, tasting, cognitiveanomalies after disease, injury (e.g. after trauma, surgical procedure,and violence), inflammatory disease of the central nervous system, suchas meningitis, encephalitis, cerebral degenerations such as Alzheimer'sdisease, Pick's disease, senile degeneration of brain, senility NOS,communicating hydrocephalus, obstructive hydrocephalus, Parkinson'sdisease including other extra pyramidal disease and abnormal movementdisorders, spinocerebellar disease, cerebellar ataxia, Marie'sSanger-Brown, Dyssynergia cerebellaris myoclonica, primary cerebellardegeneration, such as spinal muscular atrophy, familial, juvenile, adultspinal muscular atrophy, motor neuron disease, amyotrophic lateralsclerosis, motor neuron disease, progressive bulbar palsy, pseudobulbarpalsy, primary lateral sclerosis, other anterior horn cell diseases,anterior horn cell disease, unspecified, other diseases of spinal cord,syringomyelia and syringobulbia, vascular myelopathies, acute infarctionof spinal cord (embolic) (nonembolic), arterial thrombosis of spinalcord, edema of spinal cord, hematomyelia, subacute necrotic myelopathy,subacute combined degeneration of spinal cord in diseases classifiedelsewhere, myelopathy, drug-induced, radiation-induced myelitis,disorders of the autonomic nervous system, disorders of peripheralautonomic, sympathetic, parasympathetic, or vegetative system, familialdysautonomia [Riley-Day syndrome], idiopathic peripheral autonomicneuropathy, carotid sinus syncope or syndrome, cervical sympatheticdystrophy or paralysis. peripheral autonomic neuropathy in disordersclassified elsewhere, amyloidosis, diseases of the peripheral nervesystem, brachial plexus lesions, cervical rib syndrome, costoclavicularsyndrome, scalenus anticus syndrome, thoracic outlet syndrome, brachialneuritis or radiculitis NOS, including in newborn. Inflammatory andtoxic neuropathy, including acute infective polyneuritis, Guillain-Barresyndrome, Postinfectious polyneuritis, polyneuropathy in collagenvascular disease, disorders of the globe including disorders affectingmultiple structures of eye, such as purulent endophthalmitis, diseasesof the ear and mastoid process, chronic rheumatic heart disease,ischaemic heart disease, arrhythmia, diseases in the pulmonary system,respiratory system, sensoring e.g. oxygene, asthma, abnormality oforgans and soft tissues in newborn, including in the nerve system,complications of the administration of anesthetic or other sedation inlabor and delivery, diseases in the skin including infection,insufficient circulation problem, burn injury and other mechanic and/orphysical injuries, injuries, including after surgery, crushing injury,burns. Injuries to nerves and spinal cord, including division of nerve,lesion in continuity (with or without open wound), traumatic neuroma(with or without open wound), traumatic transient paralysis (with orwithout open wound), accidental puncture or laceration during medicalprocedure, injury to optic nerve and pathways, optic nerve injury,second cranial nerve, injury to optic chiasm, injury to optic pathways,injury to visual cortex, unspecified blindness, injury to other cranialnerve(s), injury to other and unspecified nerves, poisoning by drugs,medicinal and biological substances, genetic or traumatic atrophicmuscle disorders; or for the treatment of diseases or conditions ofvarious organs, such as degenerative conditions of the gonads, of thepancreas, such as diabetes mellitus type I and II, of the kidney, suchas nephrosis. Scrapie, Creutzfeldt-Jakob disease,Gerstmann-Straussler-Sheinker (GSS) disease; pain syndrome,encephalitis, drug/alcohol abuse, anxiety, postoperative nerve damage,peri-operative ischemia, inflammatory disorders with tissue damage,either by affecting the infections agent or protecting the tissue, HIV,hepatitis, and following symptoms, autoimmune disorders, such asrheumatoid arthritis, SLE, ALS, and MS. Anti-inflammatory effects,asthma and other allergic reactions, acute myocardial infarction, andother related disorders or sequel from AMI, metabolic disorders, such asobscenity lipid disorders (e.g. hyper cholestorolamia, atherosclerosis,disorders of amino-acid transport and metabolism, disorders of purineand pyrimidine metabolism and gout, bone disorders, such as fracture,osteoporosis, osteoarthritis (OA), Atrophic dermatitis, psoriasis,infection cased disorders, stem cell protection or maturation in vivo orin vitro.

Peptides used according to the invention may also be used for theprevention and treatment of achondroplasia, hypochondroplasia,platyspondylic lethal skeletal dysplasia, thanatophoric dysplasia,Antley-Bixler syndrome, Apert syndrome, Beare-Stevenson syndrome,Crouzon syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, andSaethre-Chotzen syndrome.

Antibody

It is an objective of the present invention to provide the use of anantibody, antigen binding fragment or recombinant protein thereofcapable of selectively binding to an epitope comprising a contiguousamino acid sequence derived from two distinct FGFR Ig2-NCAM F3 module1-2 binding sites or a fragment, homologue or variant thereof. Thepeptide with Seq ID NO:1 contains three a.a from the first binding siteand one a.a. from the second binding site. The peptide with Seq ID NO:2contains four a.a. from the first binding site. The peptide with Seq. IDNO:3 contains four a.a from the second binding site. The peptide withSeq ID NO:4 contains four a.a from the first binding site. The peptidewith Seq ID NO:5 contains two a.a. from the second binding site. Thepeptide with Seq ID NO:6 contains two a.a from the second binding site.The invention relates to any antibody capable of selectively binding toan epitope comprising a contiguous amino acid sequence derived from twodistinct FGFR Ig2-NCAM F3 module 1-2 binding sites, selected from any ofthe sequences set forth in SEQ ID NOS: 1-6, or a fragment or variant ofsaid sequence.

By the term “epitope” is meant the specific group of atoms (on anantigen molecule) that is recognized by (that antigen's) antibodies. Theterm “epitope” is the equivalent to the term “antigenic determinant”.The epitope may comprise 3 or more amino acid residues, such as forexample 4, 5, 6, 7, 8 amino acid residues, located in close proximity,such as within a contiguous amino acid sequence, or located in distantparts of the amino acid sequence of an antigen, but due to proteinfolding have been approached to each other.

Antibody molecules belong to a family of plasma proteins calledimmunoglobulins, whose basic building block, the immunoglobulin fold ordomain, is used in various forms in many molecules of the immune systemand other biological recognition systems. A typical immunoglobulin hasfour polypeptide chains, containing an antigen binding region known as avariable region and a non-varying region known as the constant region.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end. The constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains (Novotny J, & Haber E. ProcNatl Acad Sci USA. 82(14):4592-6, 1985).

Depending on the amino acid sequences of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are at least five (5) major classes of immunoglobulins: IgA, IgD,IgE, IgG and IgM, and several of these may be further divided intosubclasses (isotypes), e.g. IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 andIgA-2. The heavy chains constant domains that correspond to thedifferent classes of immunoglobulins are called alpha (α), delta (δ),epsilon (ε), gamma (γ) and mu (μ), respectively. The light chains ofantibodies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino sequences of their constantdomain. The subunit structures and three-dimensional configurations ofdifferent classes of immunoglobulins are well known.

The term “variable” in the context of variable domain of antibodies,refers to the fact that certain portions of the variable domains differextensively in sequence among antibodies. The variable domains are forbinding and determine the specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called complementarity determiningregions (CDRs) also known as hypervariable regions both in the lightchain and the heavy chain variable domains.

The more highly conserved portions of variable domains are called theframework (FR). The variable domains of native heavy and light chainseach comprise four FR regions, largely adopting a (3-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the p-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies. The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

An antibody that is contemplated for use in the present invention thuscan be in any of a variety of forms, including a whole immunoglobulin,an antibody fragment such as Fv, Fab, and similar fragments, a singlechain antibody which includes the variable domain complementaritydetermining regions (CDR), and the like forms, all of which fall underthe broad term “antibody”, as used herein. The present inventioncontemplates the use of any specificity of an antibody, polyclonal ormonoclonal, and is not limited to antibodies that recognize andimmunoreact with a specific antigen. In the context of both thetherapeutic and screening methods described below, preferred embodimentsare the use of an antibody or fragment thereof that is immunospecificfor an antigen or epitope of the invention.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the antigen binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Papaindigestion of antibodies produces two identical antigen bindingfragments, called the Fab fragment, each with a single antigen bindingsite, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen binding fragments that are capable of cross-linkingantigen, and a residual other fragment (which is termed pFc′).Additional fragments can include diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments. As used herein, “functional fragment” withrespect to antibodies, refers to Fv, F(ab) and F(ab′)₂ fragments.

The term “antibody fragment” is used herein interchangeably with theterm “antigen binding fragment”.

Antibody fragments may be as small as about 4 amino acids, 5 aminoacids, 6 amino acids, 7 amino acids, 9 amino acids, about 12 aminoacids, about 15 amino acids, about 17 amino acids, about 18 amino acids,about 20 amino acids, about 25 amino acids, about 30 amino acids ormore. In general, an antibody fragment of the invention can have anyupper size limit so long as it is has similar or immunologicalproperties relative to antibody that binds with specificity to anepitope comprising a peptide sequence selected from any of the sequencesidentified herein as SEQ ID NOs: 1-6, or a fragment of said sequences.Thus, in context of the present invention the term “antibody fragment”is identical to term “antigen binding fragment”.

Antibody fragments retain some ability to selectively bind with itsantigen or receptor. Some types of antibody fragments are defined asfollows:

-   -   (1) Fab is the fragment that contains a monovalent        antigen-binding fragment of an antibody molecule. A Fab fragment        can be produced by digestion of whole antibody with the enzyme        papain to yield an intact light chain and a portion of one heavy        chain.    -   (2) Fab′ is the fragment of an antibody molecule can be obtained        by treating whole antibody with pepsin, followed by reduction,        to yield an intact light chain and a portion of the heavy chain.        Two Fab′ fragments are obtained per antibody molecule.

Fab′ fragments differ from Fab fragments by the addition of a fewresidues at the carboxyl terminus of the heavy chain CH1 domainincluding one or more cysteines from the antibody hinge region.

-   -   (3) (Fab′)2 is the fragment of an antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction.    -   (4) F(ab′)₂ is a dimer of two Fab′ fragments held together by        two disulfide bonds.

Fv is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in a tight, non-covalentassociation (V_(H)-V_(L) dimer). It is in this configuration that thethree CDRs of each variable domain interact to define an antigen bindingsite on the surface of the V_(H)-V_(L) dimer. Collectively, the six CDRsconfer antigen binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

-   -   (5) Single chain antibody (“SCA”), defined as a genetically        engineered molecule containing the variable region of the light        chain, the variable region of the heavy chain, linked by a        suitable polypeptide linker as a genetically fused single chain        molecule.

Such single chain antibodies are also referred to as “single-chain Fv”or “sFv” antibody fragments. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the VH and VL domains thatenables the sFv to form the desired structure for antigen binding. For areview of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies113: 269-315 Rosenburg and Moore eds. Springer-Verlag, NY, 1994.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (VH) connected to a light chain variable domain (VL) in the samepolypeptide chain (V_(H)-V_(L)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161, and Hollinger et al., Proc. Natl.Acad Sci. USA 90: 64446448 (1993).

The invention also contemplates multivalent antibodies having at leasttwo binding domains. The binding domains may have specificity for thesame ligand or for different ligands. In one embodiment themultispecific molecule is a bispecific antibody (BsAb), which carries atleast two different binding domains, at least one of which is ofantibody origin. Multivalent antibodies may be produced by a number ofmethods. Various methods for preparing bi- or multivalent antibodies arefor example described in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175;5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

The invention contemplate both polyclonal and monoclonal antibody,antigen binding fragments and recombinant proteins thereof which arecapable of binding an epitope according to the invention.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, for example, Green et al. 1992. Production ofPolyclonal Antisera, in: Immunochemical Protocols (Manson, ed.), pages1-5 (Humana Press); Coligan, et al., Production of Polyclonal Antiserain Rabbits, Rats Mice and Hamsters, in: Current Protocols in Immunology,section 2.4.1, which are hereby incorporated by reference.

The preparation of monoclonal antibodies likewise is conventional. See,for example, Kohler & Milstein, Nature, 256:495-7 (1975); Coligan, etal., sections 2.5.1-2.6.7; and Harlow, et al., in: Antibodies: ALaboratory Manual, page 726, Cold Spring Harbor Pub. (1988), Monoclonalantibodies can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, e.g., Coligan, etal., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes, et al.,Purification of Immunoglobulin G (IgG). In: Methods in MolecularBiology, 1992, 10:79-104, Humana Press, NY.

Methods of in vitro and in vivo manipulation of monoclonal antibodiesare well known to those skilled in the art. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,1975, Nature 256, 495-7, or may be made by recombinant methods, e.g., asdescribed in U.S. Pat. No. 4,816,567. The monoclonal antibodies for usewith the present invention may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., 1991,Nature 352: 624-628, as well as in Marks et al., 1991, J Mol Biol 222:581-597. Another method involves humanizing a monoclonal antibody byrecombinant means to generate antibodies containing human specific andrecognizable sequences. See, for review, Holmes, et al., 1997, J Immunol158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma &Immunol 81:105-115.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional polyclonal antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In additional to their specificity, the monoclonal antibodiesare advantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567);Morrison et al., 1984, Proc Natl Acad Sci 81: 6851-6855.

Methods of making antibody fragments are also known in the art (see forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, NY, 1988, incorporated herein by reference). Antibodyfragments of the present invention can be prepared by proteolytichydrolysis of the antibody or by expression in E. coli of DNA encodingthe fragment. Antibody fragments can be obtained by pepsin or papaindigestion of whole antibodies conventional methods. For example,antibody fragments can be produced by enzymatic cleavage of antibodieswith pepsin to provide a 5S fragment denoted F(ab′)₂. This fragment canbe further cleaved using a thiol reducing agent, and optionally ablocking group for the sulfhydryl groups resulting from cleavage ofdisulfide linkages, to produce 3.5S Fab′ monovalent fragments.Alternatively, an enzymatic cleavage using pepsin produces twomonovalent Fab′ fragments and an Fc fragment directly. These methods aredescribed, for example, in U.S. Pat. No. 4,036,945 and U.S. Pat. No.4,331,647, and references contained therein. These patents are herebyincorporated in their entireties by reference.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody. For example, Fv fragments comprise anassociation of VH and V_(L) chains. This association may be noncovalentor the variable chains can be linked by an intermolecular disulfide bondor cross-linked by chemicals such as glutaraldehyde. Preferably, the Fvfragments comprise VH and V_(L) chains connected by a peptide linker.These single-chain antigen binding proteins (sFv) are prepared byconstructing a structural gene comprising DNA sequences encoding the VHand V_(L) domains connected by an oligonucleotide. The structural geneis inserted into an expression vector, which is subsequently introducedinto a host cell such as E. coli. The recombinant host cells synthesizea single polypeptide chain with a linker peptide bridging the two Vdomains. Methods for producing sFvs are described, for example, byWhitlow, et al., 1991, In: Methods: A Companion to Methods inEnzymology, 2:97; Bird et al., 1988, Science 242:423-426; U.S. Pat. No.4,946,778; and Pack, et al., 1993, BioTechnology 11:1271-77.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) are often involved in antigen recognition andbinding. CDR peptides can be obtained by cloning or constructing genesencoding the CDR of an antibody of interest. Such genes are prepared,for example, by using the polymerase chain reaction to synthesize thevariable region from RNA of antibody-producing cells. See, for example,Larrick, et al., Methods: a Companion to Methods in Enzymology, Vol. 2,page 106 (1991).

The invention contemplates human and humanized forms of non-human (e.g.murine) antibodies. Such humanized antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)that contain a minimal sequence derived from non-human immunoglobulin,such as the epitope recognising sequence. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a nonhuman species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. Humanized antibody(es) containing a minimalsequence(s) of antibody(es) of the invention, such as a sequence(s)recognising an epitope(s) described herein, is one of the preferredembodiments of the invention.

In some instances, Fv framework residues of the human immunoglobulin arereplaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues that are found neither in the recipientantibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, humanized antibodies will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see: Jones et al., 1986, Nature321, 522525; Reichmann et al., 1988, Nature 332, 323-329; Presta, 1992,Curr Op Struct Biol 2:593-596; Holmes et al., 1997, J Immunol158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma &Immunol 81:105-115.

The generation of antibodies may be achieved by any standard methods inthe art for producing polyclonal and monoclonal antibodies using naturalor recombinant fragments of a sequence selected from any of thesequences identified as SEQ ID NOs: 1-6, as an antigen. Such antibodiesmay be also generated using variants or fragments of SEQ ID NOs: 1-6.

The antibodies may also be produced in vivo by the individual to betreated, for example, by administering an immunogenic fragment accordingto the invention to said individual. Accordingly, the present inventionfurther relates to a vaccine comprising an immunogenic fragmentdescribed above.

The application also relates to a method for producing an antibody ofthe invention said method comprising a step of providing of animmunogenic fragment described above.

The invention relates both to an antibody, which is capable ofmodulating, such as enhancing or attenuating, biological function ofNCAM in particular a function related to neural cell growth andsurvival, and to an antibody, which can recognise and specifically bindto NCAM without modulating biological activity thereof.

The invention relates to use of the above antibodies for therapeuticapplications involving the modulation of activity of NCAM

In one aspect the invention relates to the use of a pharmaceuticalcomposition comprising an antibody described above.

EXAMPLES 1. Binding Between FGFR Ig2 and NCAM F3 Module 1-2 Methods

To study the binding properties between the FGFR Ig2 module and NCAM, weused recombinant proteins of the NCAM F3 modules 1-2 and the FGFR1 Ig2module. Both proteins were properly folded as judged by one-dimensionalNMR.

Production of Recombinant Proteins

The Ig2 module of mouse FGFR1 (IIIc isoform) and rat NCAM F3 modules 1-2were produced. Both proteins were expressed in a KM71 strain of yeastexpression system of P. pastoris (Invitrogen, USA). The FGFR Ig module 2consists of AGHHHHHH and amino acids 140-251 of FGFR (SwissProt p16092)and the combined F3 modules 1 and 2 consist of AGHHHHHH and amino acids507-611 and 507-705 of NCAM (SwissProt p13596), respectively. For theprotein expression the following media were used: BMGH (0.1 M potassiumphosphate pH 6.0, 3.4 g/L yeast nitrogen base (without amino acids andammonium sulphate), 10 g/L ammonium sulphate, 400 pg/L D-biotin, 60 mg/LL-histidine, 2% glycerol) and BMMH (0.1 M potassium phosphate pH 6.0,3.4 g/L yeast nitrogen base (without amino acids and ammonium sulphate),10 g/L ammonium sulphate, 400 [ig/L D-biotin, 60 mg/L L-histidine, 1%methanol). For production of the ¹⁵N labelled protein, ¹⁵N labelledammonium sulphate was used in the media in the following concentrations:3.5 g/L in BMGH and 1.5 g/L in BMMH. All the proteins were purified byaffinity chromatography using Ni²+-NTA resin (Qiagen) and/or ionexchange chromatography and gel filtration.

NMR Analysis

The following samples were used recording of NMR spectra: 10 p.M ¹⁵NFGFR Ig2 module with or without 13, 25, 40 μM NCAM F3 modules 1-2. Thebuffer was 10 mM sodium phosphate containing 150 mM NaCl, pH 7.4. Thesamples were analyzed by recording a ¹⁵N-Heteronuclear single quantumcorrelation (HSQC) spectra using the standard set-up provided byProteinPack. HSQC spectrum of a ¹⁵N-labeled protein records the one-bondcoupling of an H—N bond and is therefore a useful tool for monitoringsite-specific perturbations. The chemical shift changes of the signalsprovide a means to identify the amino acid residues whose NMR signalsare perturbed by the binding of another molecule. The addition of F3NCAM modules led to either line-broadening, chemical shift changes or,for certain residues, disappearance of the NMR signals. The residueswith NMR signal undergoing significant chemical shift changes (greaterthan 0.03 ppm for the 13 μM F3 module and 0.05 ppm for the 25 and 40 μMF3 modules) or disappearing completely were considered to bespecifically perturbed by the binding. The spectra were processed byNMRPipe (Delaglio et al., 1995) and analysed by Pronto3D (Kjaer et al.,1994). The NMR experiments were performed using Varian Unity Inova 750and 800 MHz spectrometers. All spectra were recorded at 298 K.

SPR Analysis

Immobilization of the NCAM and FGFR modules and binding analysis wereperformed using a BIAcore2000 instrument (Biosensor AB, Sweden) at 25°C. using 10 mM sodium phosphate containing 150 mM NaCl (pH 7.4) asrunning buffer. Data were analyzed by nonlinear curve fitting using themanufacturer's software. The FGFR Ig module 2 and NCAM F3 modules 1-2were immobilized on sensor chip CM5 using an amine coupling kit(Biosensor AB) as follows: the chip was activated by 20 μl activationsolution; the protein was immobilized using 12 μl of 20 μg/ml protein in10 mM sodium acetate buffer (pH 6.0); the chip was blocked by 35 μlblocking solution. The curves corresponding to the difference betweenbinding to ligand (immobilized molecule) and a blank chip were used foranalysis.

Results and Discussion Binding of FGFR Ig2 to NCAM

It has previously been shown by SPR that the double-module construct ofNCAM F3 modules 1-2 binds to the double-module FGFR Ig2-Ig3 constructwith a dissociation constant (Kd) of 10 μM (Kiselyov et al., 2003).However, interaction between the individual modules could hardly bedetected by SPR. This indicates that both modules of the NCAM and FGFRconstructs are involved in the binding, or are necessary for thefull-strength binding. Using a more sensitive method (NMR), aninteraction between the NCAM F3 module 2 and FGFR Ig3 module has beendetected (the binding between the NCAM F3 module 1 and FGFR Ig2 modulewas not tested). The binding site in the NCAM F3 module 2 was mapped tothe FG-loop region of the module, which is located in the module'sN-terminus (Kiselyov et al., 2003). This indicates that the C-terminusof the NCAM F3 module 1 together with the N-terminal part of the NCAM F3module 2 could form a single binding site for FGFR, which may bedestroyed when the modules are separated.

Binding of the FGFR Ig2 module to the combined NCAM F3 modules 1-2 wastested. Using SPR, binding of soluble Ig2 to immobilized NCAM F3 modules1-2 was detected with an estimated KD value of 35 μM (see FIG. 1A). Whenthe Ig2 module was immobilized, the KD value for the binding of solubleF3 modules 1-2 was estimated to be 12 μNA (FIG. 1B). These data showthat the Ig2 module of FGFR is also involved in binding to NCAM F3modules.

Identification of the FGFR Ig2 Residues Involved in Binding to NCAM

Since the Ig2 module of FGFR binds to NCAM F3 modules 1-2, it was ofinterest to determine the residues of the Ig2 module involved in thisbinding. For this purpose, we used NMR analysis. In a ¹⁵N—HSQC spectrumof a ¹⁵N-labeled protein, a signal for all amino acids with both anitrogen and a proton can be observed. The changes in chemical shifts ofthe signals provide a method for identification in a protein of aminoacid residues that are perturbed by the binding of another molecule. Thespectrum of 10 μM Ig2 module was recorded in the presence of 0, 13, 25or 40 RM F3 modules 1-2. Addition of the NCAM F3 modules led tobroadening of the resonance lines, changes of the chemical shifts anddisappearance of the NMR signals for some of the residues. The residueswith significant changes of the chemical shifts (greater than 0.03 ppmfor 13 μM and 0.05 ppm for 25 and 40 OA F3 modules) or with thecompletely disappeared NMR signals were considered to be significantlyperturbed by the binding. The recorded changes of chemical shifts andmapping of the significantly perturbed residues onto the structure ofthe Ig2 module (Plotnikov et al., 1999) are shown in FIG. 2. As can beseen from FIG. 2C, addition of 40 μNA F3 modules perturbed most of theIg2 module's residues and most of these residues are those with thedisappeared signals. This indicates that the exchange between the boundand non-bound form of the Ig2 module is intermediate on the NMRtime-scale. Addition of 13 μM F3 modules led to perturbation of fewresidues (FIG. 2A), while 25 μM F3 modules perturbed 28 residues whichform two clusters on the opposite ‘sides’ of the module (FIG. 2B). Thefirst binding site comprises a cluster involving residues T¹⁵⁶, S¹⁵⁷,E¹⁵⁹, A¹⁷¹, T¹⁷³, V¹⁷⁴, K¹⁷⁵, S¹⁸¹, S²¹⁴, M²¹⁷, D²¹⁸, S²¹⁹ and V²²⁰,from the region a.a 140-251 of FGFR corresponding to FGFR Ig2, and thesecond binding site comprises a cluster involving residues M¹⁶¹, L¹⁹¹,K¹⁹², N¹⁹³, F¹⁹⁷, V²²¹, T²²⁹, C²³⁰, D²⁴⁶ and V²⁴⁸, from the region a.a140-251 of FGFR corresponding to FGFR Ig2. Perturbation of theseresidues demonstrates that the presence of the NCAM F3 modules close tothe FGFR Ig2 module alters the chemical environment at the perturbedresidues, indicating that the perturbed residues are either a part or inthe vicinity of the binding site for the interaction between NCAM andFGFR.

One of the identified clusters (the first one) is located in the closevicinity of the sites in Ig2 for binding of FGF, heparin and Ig2 (FIG.3) (Plotnikov et al., 1999; Pellegrini et al., 2000). As can be seenfrom FIG. 3, heparin is expected to inhibit binding of the Ig2 module toNCAM. To verify this assumption, we tested sucrose octasulphate (SOS), awell-known heparin analogue, for its ability to inhibit binding ofsoluble Ig2 to the immobilized NCAM F3 modules. As appears from FIG. 4,SOS was indeed capable of inhibiting the Ig2-NCAM binding, as expectedgiven that the first cluster is located close to the heparin-bindingsite of the Ig2 module.

We suggest that in the absence of NCAM mediated cell-cell adhesion, thatis when NCAM is not involved in the homophilic binding, the FGFRmolecules do not bind substantially to NCAM. However, when NCAM isinvolved in homophilic binding, the NCAM molecules become clustered dueto formation of zipper-like structures. This would allow an FGFRmolecule to bind simultaneously to two neighbouring NCAM molecules witha much higher affinity than to the individual NCAM molecules, which issupposed to ensure an efficient NCAM-FGFR interaction. Thus FGFR isexpected to bind to and become activated by NCAM only when NCAM isclustered through a homophilic binding mechanism.

REFERENCES

-   Atkins A R, Osborne M J, Lashuel H A, Edelman G M, Wright P E,    Cunningham B A, Dyson H J (1999). Association between the first two    immunoglobulin-like domains of the neural cell adhesion molecule    N-CAM. FEBS Lett. 451:162-168.-   Berezin V, Bock E and Poulsen F M (2000). The neural cell adhesion    molecule. Current Opinion in Drug Discovery & Development 3:605-609.-   Bruses J L, Rutishauser U (2001). Roles, regulation, and mechanism    of polysialic acid function during neural development. Biochemie    83:635-643.-   Cremer H, Chazal G, Goridis C, Represa A (1997). NCAM is essential    for axonal growth and fasciculation in the hippocampus. Mol Cell    Neurosci. 8:323-335.-   Delaglio F, Grzesiek S, Vuister G W, Zhu G, Pfeifer J and Bax A    (1995). NMRPipe: a multidimensional spectral processing system based    on UNIX pipes. J Biomol NMF? 6:277-293.-   Doherty P and Walsh F S (1996). CAM-FGF Receptor Interactions: A    Model for Axonal Growth. Mol Cell Neurosci. 8:99-111.-   Itoh N and Ornitz D M (2004). Evolution of the Fgf and Fgfr gene    families. Trends Genet. 20:563-569.-   Jensen P H, Soroka V, Thomsen N K, Ralets I, Berezin V, Bock E,    Poulsen F M (1999). Structure and interactions of NCAM modules 1 and    2, basic elements in neural cell adhesion. Nat Struct Biol.    6:486-493.-   Kasper C, Rasmussen H, Kastrup J S, Ikemizu S, Jones E Y, Berezin V,    Bock E, Larsen I K (2000). Structural basis of cell-cell adhesion by    NCAM. Nat Struct Biol. 7:389-393.-   Kiselyov V V, Berezin V, Maar T E, Soroka V, Edvardsen K, Schousboe    A, Bock E (1997). The first immunoglobulin-like neural cell adhesion    molecule (NCAM) domain is involved in double-reciprocal interaction    with the second immunoglobulin-like NCAM domain and in heparin    binding. J Biol Chem. 272:10125-10134.-   Kiselyov V V, Kochoyan A, Poulsen F M, Bock E, Berezin V (2006b).    Elucidation of the mechanism of the regulatory function of the Ig1    module of the fibroblast growth factor receptor 1. Protein Sci.    15:2318-2322.-   Kiselyov V V, Bock E, Berezin V, Poulsen F M (2005). Structural    biology of NCAM homophilic binding and activation of FGFR. J    Neurochem. 94:1169-1179.-   Kiselyov V V, Skladchikova G, Hinsby A M, Jensen P H, Kulahin N,    Soroka V, Pedersen N, Tsetlin V, Poulsen F M, Berezin V and Bock E    (2003). Structural basis for a direct interaction between FGFR1 and    NCAM and evidence for a regulatory role of ATP. Structure    11:691-701.-   Kiselyov V V, Bock E, Berezin V, Poulsen F M (2006a). NMR Structure    of the First Ig Module of mouse FGFR1. Protein Sci. 15:1512-1515.-   Kjaer M, Andersen K V and Poulsen F M (1994). Meth Enzymol.    239:288-307.-   McKeehan W L, Wang F, Kan M (1998). The heparin sulphate-fibroblast    growth factor family: diversity of structure and function. Prog    Nucleic Acid Res Mol Biol 59:135-176.-   Olsen S K, Ibrahimi O A, Raucci A, Zhang F, Eliseenkova A V, Yayon    A, Basilico C, Linhardt R J, Schlessinger J and Mohammadi M (2004).    Insights into the molecular basis for fibroblast growth factor    receptor autoinhibition and ligand-binding promiscuity. Proc Natl    Acad Sci USA 101:935-940.-   Pellegrini L, Burke D F, von Delft F, Mulloy B and Blundell T L    (2000). Crystal structure of fibroblast growth factor receptor    ectodomain bound to ligand and heparin. Nature 407:1029-1034.-   Plotnikov A N, Schlessinger J, Hubbard S R and Mohammadi M (1999).    Structural basis for FGF receptor dimerization and activation. Cell    98:641-650.-   Rougon G, Hobert 0 (2003). New insights into the diversity and    function of neuronal immunoglobulin superfamily molecules. Annu Rev    Neurosci. 26:207-238.-   Soroka V, Kolkova K, Kastrup J S, Diederichs K, Breed J, Kiselyov V    V, Poulsen F M, Larsen I K, Welte W, Berezin V, Bock E, Kasper C    (2003). Structure and interactions of NCAM Ig1-2-3 suggest a novel    zipper mechanism for homophilic adhesion. Structure 11:1291-1301.-   Walmod P S, Kolkova K, Berezin V, Bock E (2004). Zippers make    signals: NCAM-mediated molecular interactions and signal    transduction. Neurochem Res. 29:20152035.-   Wang F, Kan M, Yan G, Xu J and McKeehan W L (1995). Alternately    spliced NH2-terminal immunoglobulin-like Loop I in the ectodomain of    the fibroblast growth factor (FGF) receptor 1 lowers affinity for    both heparin and FGF-1. J Biol Chem. 270:1023110235.

1.-35. (canceled)
 36. A method for modulating NCAM signaling comprisingadministering to a subject in need thereof an isolated peptide of 5 to25 contiguous amino acids, said peptide comprising an amino acidsequence derived from FGFR Ig2 and selected from the group consistingof: (SEQ ID NO: 1) TSPEKMEKKL (SEQ ID NO: 2) AKTVKFK (SEQ ID NO: 3)RWLKNGKEFK (SEQ ID NO: 4) TWSIIMDSV (SEQ ID NO: 5) SDKGNYTCIVEN and(SEQ ID NO: 6) TYQLDVVERS,

or a fragment or variant thereof, said variant being at least 70%identical to said amino acid sequence, and said fragment having a lengthof 5 or more amino acid residues, wherein said peptide specificallybinds to NCAM.
 37. The method according to claim 36, wherein saidpeptide is in monomeric form.
 38. The method according to claim 36,wherein said peptide is in multimeric form.
 39. The method according toclaim 36, wherein said variant is at least 80% identical to said aminoacid sequence.
 40. The method according to claim 36, wherein saidpeptide is 5 to 15 amino acids in length.
 41. The method according toclaim 36, wherein said peptide specifically binds to the NCAMfibronectin 3, module 1 or
 2. 42. The method according to claim 36,wherein said peptide activates NCAM signaling.
 43. The method accordingto claim 36, wherein said peptide induces neurite outgrowth and/orpromotes neural cell survival and/or stimulates synaptic plasticityand/or stimulates learning and/or memory including short term and longterm memory.
 44. The method according to claim 36, wherein said peptidecomprises the amino acid sequence of SEQ ID NO:2, or a fragment havingat least 5 consecutive amino acids of SEQ ID NO:2.
 45. The methodaccording to claim 36, wherein said peptide comprises the amino acidsequence of SEQ ID NO:3, or a fragment having at least 5 consecutiveamino acids of SEQ ID NO:3.
 46. The method according to claim 36,wherein said peptide comprises or consists of the amino acid sequence ofSEQ ID NO:2, or a variant having at least 80% identity to SEQ ID NO:2.47. The method according to claim 36, wherein said peptide comprises orconsists of the amino acid sequence of SEQ ID NO:3, or a variant havingat least 70% identity to SEQ ID NO:3.
 48. The method according to claim36, wherein said peptide comprises or consists of the amino acidsequence of SEQ ID NO:3, or a variant having at least 80% identity toSEQ ID NO:3.
 49. The method according to claim 36 wherein the subjecthas a disease or condition of the central nervous system or theperipheral nervous system.
 50. The method according to claim 36 whereinthe subject has a condition selected from the group consisting ofpostoperative nerve damage, traumatic nerve damage, impaired myelinationof nerve fibers, post-ischaemic damage, multi-infarct dementia, multiplesclerosis, nerve degeneration associated with diabetes mellitus,neuromuscular degeneration, schizophrenia, mood disorders, manicdepressive disorders, prion diseases, cancer, Alzheimer's disease,Parkinson's disease, and Huntington's disease.
 51. The method accordingto claim 36 wherein the subject has a disease or condition of themuscles, or a disease or degenerative condition of the gonads, of thepancreas, of the kidney or of the heart.
 52. The method according toclaim 36 wherein the subject has acute myocardial infarction.
 53. Themethod according to claim 36 wherein the subject has a wound and whereinthe peptide promotes wound healing.
 54. A pharmaceutical compositioncomprising an isolated peptide as defined in claim
 36. 55. A method formodulating NCAM signaling comprising administering to a subject in needthereof an isolated peptide consisting of an amino acid sequence derivedfrom FGFR Ig2 and selected from the group consisting of: (SEQ ID NO: 1)TSPEKMEKKL (SEQ ID NO: 2) AKTVKFK (SEQ ID NO: 3) RWLKNGKEFK(SEQ ID NO: 4) TWSIIMDSV (SEQ ID NO: 5) SDKGNYTCIVEN and (SEQ ID NO: 6)TYQLDVVERS

or a fragment or variant thereof, said variant being at least 70%identical to said amino acid sequence, and said fragment having a lengthof 5 or more amino acid residues, wherein said peptide specificallybinds to NCAM.